(This tip relates to Skyline v 4.2 or later.)

This is a quick demonstration on how to use Skyline to generate an overlapping-window isolation window list suitable for acquisition using the approach described in this manuscript and downstream computational demultiplexing.

To begin this demo, start with a blank Skyline document: 

• From the Skyline landing page, click Blank Document

• On the Settings menu, click Transition Settings and in the Transition Settings dialog box, click the Full Scan tab.

• Under MS/MS filtering, click the Acquisition method dropdown and select DIA. 
• For the Isolation scheme, select Add ...

• In the Edit Isolation Scheme dialog, enter a name for the Isolation scheme such as '20mz_overlap' and then click the Prespecified isolation windows radio button.  

• Click the Calculate ... option and enter parameters for the method.  
• Enter '500' for Start m/z.
• End m/z is '500'.
• Window width should be '20'.
• Be sure that Deconvolution is set to Operlap and that Optimize Window Placement is checked.

• Click OK to close the Calculate Isolation Scheme dialog and OK to close the Edit Islation Scheme window.
• Click OK once again to close the Transition Settings dialog.
• From the main Skyline menu, select File and then Export.  From the flyout menu, click the Isolation List ... option. 

• In the Export Isolation List window, select the appropriate instrument (for this example, 'Thermo Q Exactive') and choose a file location in which to save the list of isolation windows.

The resulting isolation list contains the isolation centers of each isolation window in the order that they should be acquired. NOTE: the isolation centers will need to be regenerated using this approach if any of the other acquisition parameters such as isolation width or m/z range covered are changed.